1. Field of the Invention
The present invention relates to encapsulated living cells, their preparation and uses.
2. Brief Description of Prior Art
The use of cells, either of animal or vegetable origin, in vitro, has recently received renewed attention due to a number of important technological developments. For example, in vitro cell cultures of hybridomas are now routinely utilized for the preparation of monoclonal antibodies of great specificity. Cancer cell lines are utilized in vitro for formation of such hybridomas, and also for the screening and testing of potential carcinogenic and anticarcinogenic compounds. Pancreatic cells have been utilized in vitro and in vivo for the production and delivery of insulin. Also, the industrial utilization of isolated immobilized cells has received attention, since these can be used as catalysts for biochemical reactions, and such reactions can be used as important tools in syntheses and analytical determinations (see, for example, Venkatsubramanian, "Immobilized Microbial Cells," 106 ACS Symposium Series, (1979)).
In many instances, the direct introduction of a foreign cell into a host can produce severe immune responses in the host. For example, when growing hybridoma cells in the ascites fluid of a host such as a mouse, the mouse has to be pretreated so as to prevent immune response. When injecting whole insulin cells into a human, immune response is also a complicating feature. A need, therefore, exists for improved methods of facilitating the introduction of such cells into a host, as well as generally, for facilitating the manipulation of cells in vitro.
A number of methods has been described in the prior art relating to either the encapsulation or entrapment of biologically active materials, albeit not of living cells.
Lim et al, for example, in U.S. Pat. Nos. 4,251,387, 4,255,411 and 4,257,884, describe techniques for producing semi-permeable microcapsules by interfacial polymerization, and their uses in immunoassays and chromatography. The material to be encapsulated and a hydrophilic monomer are emulsified in a hydrophobic continuous phase. Polymerization is initiated by dissolving a second monomer in the continuous phase, and occurs only at the interface between the hydrophilic droplets and hydrophobic continuous phase, to result in the formation of macroporous, poorly-defined capsule membranes. The affinity of the continuous phase for the hydrophilic monomer is varied by altering the polarity of the continuous phase, and microcapsules having uniform capsule membranes and selected upper limits of permeability can be produced. Balassa, U.S. Pat. No. 3,780,195, describes a process for encapsulating active materials in a shell composition in which the capsule composition is formed by dispersing an active material and a shell composition in a solvent for the shell composition. The capsule composition is formed into particles containing the active material in a dispersed phase, and removing the solvent from the shell composition solution by adding a lower molecular weight polyglycol. Preferably the desolventizing operation can be accelerated by first dispersing the capsule composition in a viscous white mineral oil to form discrete particles, and then admixing the mixture with anhydrous polyglycol. Among the shell materials are included proteins, such as egg and blood albumin. Other patents dealing with the encapsulation of medicaments or (non-living) natural products, are Valentine et al, U.S. Pat. No. 2,889,252, Kitajima et al, U.S. Pat. Nos. 3,691,090 and 3,714,065; Scarpelli, U.S. Pat. No. 3,516,942; Pasin, U.S. Pat. No., 3,664,963; Ogawa, U.S. Pat. No. 3,642,978 and Soloway, U.S. Pat. No. 3,137,631. The Soloway patent discloses encapsulation in natural products such as albumins, followed by treatment with crosslinking agents such as formaldehyde, glyoxal and the like, in order to increase the stability of the capsule walls.
Among patents which describe the formation of soft solid microparticles, not capsules, having homogeneously dispersed therein various medicaments and therapeutic compositions are Meeks et al, U.S. Pat. No. 4,187,285, (technetium-99m dispersed in albumin); Yapel, Jr., U.S. Pat. No. 4,147,767 (drugs dispersed in solid serum albumin); Oppenheim et al, U.S. Pat. No. 4,107,288 (drugs dispersed in particles, including serum albumin particles, which are crosslinked); Zolle, U.S. Pat. No. 3,937,668 (solid albumin particles carrying radioactivity, drugs, insecticides, dyes and the like); Winchell et al, U.S. Pat. No. 4,024,233 (microaggregate human serum albumin having dispersed tin); and Layne et al, U.S. Pat. No. 4,094,965 (tin and a radionuclide dispersed in albumin).
A system for the microencapsulation of a great variety of substances, including antigens of various microorganisms such as bacteria and viruses, is described in copending U.S. application Ser. No. 194,127, filed Oct. 6, 1980, at the U.S. Patent and Trademark Office, for "Microencapsulation Process" by Tice and Lewis, now U.S. Pat. No. 4,389,330. This application is herein incorporated by reference. The microencapsulation process taught in this application comprises dissolving or dispersing an active agent in a solvent, and dissolving wall-forming material in said solvent; dispersing the solvent containing the active agent and wall-forming material in a continuous phase processing medium; evaporating a portion of the solvent from the dispersion step, thereby forming microcapsules containing the active agent in the suspension; and finally extracting the remainder of the solvent from the microcapsules.
The problems confronted by the practitioner in attempting to extend all of these prior art techniques to the encapsulation of living cells, however, are multiple. Many of the techniques described in the prior art operate under conditions which are too drastic for the survival or continuing viability of a living cell. For example, the use of organic solvents, high temperatures, reactive monomers, crosslinking conditions, and the like may hamper the survival of cells intended to be encapsulated. Moreover, it is crucial to prevent dehydration or osmotic rupture of the cell. Another serious problem is the necessity of providing the microcapsule walls with sufficient permeability for nutrients and excretion products Along the same lines, if the cell is used as a source of macromolecules or biological assemblies, such as antibodies or virions, respectively, it is necessary to assure the existence of pores of sufficient size to permit the exit of such macromolecules. If the cells are encapsulated and injected into a host so that they would provide a continuous source of macromolecules or biological assemblies, the pores have to be of the right diameter to allow the transport of the macromolecules or assemblies to the extra capsular medium, yet prevent the entry of molecules or cells of a host which would destroy the encapsulated cells due to the immune system of the host.
A need, therefore, continues to exist for a method to encapsulate living cells under sufficient mild conditions which would allow the living cells to retain viability yet will also allow the formation of a controlled porosity in the capsule walls.